Annual seasonal outbreaks of Influenza A virus (IAV) cause serious public health and economic concerns, and the potential for pandemics resulting in disease of increased severity, morbidity and mortality necessitates the study of factors that enable IAV to establish an infection in a na?ve host. Frequently, viruses with pandemic potential are those that replicate in an avian or swine host normally and have acquired to ability to infect and transmit between humans with no prior exposure to the pathogen. The process of adaptation to and transmitting between new hosts is multifactorial, however the hemagglutinin (HA) protein, responsible for receptor binding and fusion of the viral and host membranes during entry, plays a significant role. It is known that the conformation of sialic acid, the substrate fo HA, is a determinant of species tropism for the virus. Avian viruses recognize and bind, via the HA, receptors that terminate in ?2,3 linked sialic acid. Human viruses recognize and bind receptors that terminate in ?-2,6 linked terminal sialic acid. The sialic acid specificity of the A is considered to be a factor in the restriction of direct transmission of avian viruses to humans. The neuraminidase (NA) protein of IAV is a sialidase and is thought to clear the sialic acids, from mucins lining the airway and from the surface of already infected cells, so that the virus may bud out and disseminate to infect other nearby cells. However, the role of NA prior to infection has not been as extensively characterized. The HA and NA share a substrate, so it has been proposed that a functional balance between the two proteins must exist in order for the virus to maintain a productive infection. We postulate that not only does the sialidase of the NA match the binding affinity of the HA as suggested by others, but also that the sialic acid cleavage specificity of NA balances the sialic acid binding specificity of HA. It is possible that the NA acs on the cell surface glycans of an uninfected host cell prior to sustained HA binding and removes certain structures, therefore limiting the range of potential receptors for HA. We propose to use glycan microarray technology to determine the specificity of a range of NAs and the effect of NA activity on the binding profiles of HA. We will also examine the affects of adaptation to a new host species on the HA and NA, by passaging recombinant viruses with the same internal genes expressing the HA and NA proteins of a range of subtypes in different cell culture systems, including embryonated chicken eggs, swine and human primary epithelial cells, and Madin- Darby canine kidney cells. We expect to see changes in both proteins reflecting the necessity of recognition of a different sialic acid linkage conformation. We will extend our studies to examine the co-evolution of the HA and NA to establish a functional balance by serially passaging recombinant viruses containing non-cognate HA:NA pairs in the same cell culture systems. The findings of the proposed studies will expand our knowledge of the role of NA prior to receptor binding and entry and of the process of adaptation to a new host, with implications for the role of HA and NA and their functional balance in the event of cross-species transmission.